In eukaryotes, entry into mitosis is regulated by the synergistic and opposing activities of a cascade of distinct protein kinases and phosphatases. This cascade converges on CDC2, a serine/threonine protein kinase required for the entry of cells into mitosis. Two neighboring amino acids within the N-terminus of CDC2 (threonine 14 and tyrosine 15) have been shown to be critical elements in regulating the kinase activity of CDC2. Phosphorylation of these residues quantatively inhibits the activity of the CDC2 kinase and thereby prevents cells from entering into mitosis. The goals of this grant are to characterize the kinases that either directly catalyze or indirectly regulate phosphorylation of these key residues. cDNAs encoding the human and Xenopus Thr14/Tyr 15 kinases will be isolated. Specific probes will be generated and each kinase will be characterized both in vitro and in vivo. In addition, upstream regulators of these enzymes will be identified. In fission yeast, the wee1, nim1/cdr1 and mik1 gene products will be studied. Finally, the optimal peptide motif for each kinase will be determined in order to identify potential substrates of the kinases (other that CDC2) and to develop specific peptide inhibitors for each kinase. These inhibitors will be used as in vivo probes of protein function and they may also serve as templates for the design of novel drugs targeted to diseases that arise as a result of aberrant cellular proliferation.